Project Summary Our objective is to produce Lactococcus lactis-based nanobody-expressing strains and demonstrate that the nanobodies produced can effectively neutralize Clostridium difficile toxins in vitro and in vivo. C. difficile is a ubiquitous anaerobic gram-positive bacterium that can sporulate and become highly resistant to environment stresses and frontline antibiotics such as clindamycin1. Pathogenic strains of C. difficile secrete varying combinations of virulent glycosyltransferases, the large clostridial toxins A (TcdA) and B (TcdB), and binary toxin, C. difficile transferase (CDT)2. TcdA and TcdB disrupt tight junctions and enter the lamina propria to promote inflammation. CDT synergizes with TcdA and TcdB to worsen C. difficile infection (CDI), inducing cell protrusions that increase adherence of C. difficile, and suppressing protective host eosinophilic response in the colon3. The toxin enzymatic activities damage intestinal epithelial cells, resulting in inflammatory colitis, severe diarrhea, abdominal pain, flu-like symptoms, and even death1. CDI often occurs following antibiotic-treatment when the endogenous microflora of individuals is altered or severely reduced. While existing antibiotics are effective in many patients, continuous antibiotic usage further damages the microbiota contributing to recurring CDI. As a result, recurrent CDI is currently most effectively treated with fecal microbiota transplant (FMT) therapy4-6. FMT, however, is an expensive, heterogeneous and poorly defined therapeutic that requires special FDA approval due to safety concerns7. As a result, new approaches are desperately needed to prevent and treat CDI. We recently generated unique nanobodies from a camelid VHH phage display. Our studies demonstrate that the identified nanobodies bind to key conformational epitopes of clostridial toxins A (TcdA), B (TcdB), and C. difficile transferase/binary toxin (CDT), with the potential to neutralize CDI-associated toxins in the intestinal tract. To orally deliver these nanobodies into the intestinal tract and improve mucosal pharmacokinetics/pharmacodynamics following oral administration, our strategy is to express the nanobodies in the context of L. lactis. We believe L. lactis represents an ideal delivery vehicle as it has received ?Generally Regarded as Safe? status from the FDA and does not colonize in the human gastrointestinal tract. This proposal is designed to validate a novel L. lactis-based nanobody platform for rapid development and delivery of nanobodies into the intestinal tract. At the same time, we will validate the first drug candidate using this platform for the prevention and treatment of CDI. The specific aims are to: 1) Select therapeutic nanobodies based on domain mapping and C. difficile toxin neutralization, 2) Generate L. lactis strains that express a neutralizing nanobody against either TcdA, TcdB, or CDT, and 3) Demonstrate that combined strains inhibit C. difficile pathogenesis in a murine model.